The production of tobacco with decreased levels of nicotine is of interest, given concerns regarding the addictive nature of nicotine. Additionally, tobacco plants with extremely low levels of nicotine production, or no nicotine production, are attractive as recipients for transgenes expressing commercially valuable products such as pharmaceuticals, cosmetic components, or food additives. Various processes have been designed for the removal of nicotine from tobacco. However, most of these processes remove other ingredients from tobacco in addition to nicotine, thereby adversely affecting the tobacco. Classical crop breeding techniques have produced tobacco plants with lower levels of nicotine (approximately 8%) than that found in wild-type tobacco plants. Tobacco plants and tobacco having, even further reductions in nicotine content are desirable.
Nicotine is formed primarily, in the roots of the tobacco, plant and is subsequently transported to the leaves, where it is stored (Tso, Physiology and Biochemistry of Tobacco Plants, pp. 233–34, Dowden, Hutchinson & Ross, Stroudsburg, Pa. (1972)). Nicotine is produced by the condensation of two precursors, nicotinic acid and N-methylpyrolinium, that arise from two separate biosynthetic pathways (see FIG. 1)(Bush and Saunders (1977) Proc. Am. Chem. Soc. Symp., New Orleans, pp. 389–425; Hashimoto and Yamada (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 257–285; Waller and Dermer (1981) In: The Biochemistry of Plants: A Comprehensive Treatise, P. K. Stumpf and E. E. Conn, eds. Academia Press, pp. 317–395). The pyridine nucleotide cycle synthesize nicotinic acid (Wagner et al. (1986) Planta 167, 226–232; Wagner and Wagner (1985) Planta 165, 532–537), whereas N-methylpyrrolinium cations are synthesized from ornithine or arginine via putrescence (Leete (1980) In: Encyclopedia of Plant Physiology, Secondary Plant Products, Vol. 8, E. A. Bell and B. V. Charlwood, eds, Springer-Verlag, pp. 65–91; Tiburcio and Galston (1986) Phytochemistry, 25, 107–110). Reciprocal grafting experiments have demonstrated that nicotine is synthesized in roots and transported through the xylem to leaves and other plant organs (Dawson (1941) Science, 94, 396397).
Two regulatory loci (Nic1 and Nic2) regulate nicotine production. Legg et al. ((1969) J. Hered, 60, 213–217) incorporated genes from low alkaloid content Cuban cigar cultivars into Burley 21 cultivars. These investigators showed that the low alkaloid lines differed from standard cultivars at two loci, Nic1 (formerly identified as A) and Nic2 (formerly identified as B). These two loci are unlinked and the gene action is semi-dominant and primarily additive (Legg et al. (1969) J. Hered, 60, 213–217). Collins et al. ((1974) Crop Sci., 14, 77–80) prepared doubled haploid tobacco breeding lines of these four alkaloid genotypes. The genotype of standard cultivars is Nic1/Nic1 Nic2/Nic2 and that of low nicotine lines is nic1/nic1 nic2/nic2. Nic1/Nic1 nic2/nic2 is a high intermediate and nic1/nic1 Nic2/Nic2 is a low intermediate (Legg and Collins (1971) Can. J. Genet. Cytol. 13, 287–291). These lines are similar in days-to-flower, number of leaves, leaf size, and plant height. Enzyme analyses of roots of single and double Nic mutants show that the activities of two enzymes, quinolinate phosphoriboxyl transferase (QPTase) and putrescence methyl transferase (PMTase), are directly proportional to levels of nicotine biosynthesis (Saunders and Bush (1979) Plant Physiol 64:236). Both Nic1 and Nic2 affect PMTase and QPTase activities in roots, and thus, regulate nicotine synthesis (Leete (1983) In: Alkaloids: Chemical and Biological Perspectives, S. W. Pelletier, ed. John Wiley & Sons, pp. 85–152).
Hibi et al. ((1994) Plant Cell, 6, 723–735) isolated the cDNA encoding PMTase, PMT, and showed that PMT transcript levels are regulated by Nic1 and Nic2. The QPTase cDNA and genomic clones (Nt{tilde under (O)}PT1) have also been isolated and the transcript levels of Nt{tilde under (O)}PT1 are also regulated by Nic1 and Nic2 (Song, W., Mendu, N., and Conkling, M. A. (1999) Plant Cell, in preparation). Thus, it appears that the Nic genes regulate nicotine content by regulating the transcript levels of genes encoding the two rate-limiting enzymes, PMTase and QPTase. Further, Nic1 and Nic2 have been shown to be positive regulators of Nt{tilde under (O)}PT1 transcription and that promoter sequences upstream of the transcription initiation site contain the cis-acting sequences necessary for Nic gene product activation of Nt{tilde under (O)}PT1 transcription. Because expression of QPTase and PMTase are coordinately-regulated by the Nic gene products, it likely that the Nic gene products also directly regulate transcription of the PMT gene.
One approach for reducing the level of a biological product, such as nicotine, is to reduce the amount of a required enzyme (i.e. QPTase and PMTase) in the biosynthetic pathway leading to that product. Where the affected enzyme naturally occurs in a rate-limiting amount (relative to the other enzymes required in the pathway), any reduction in that enzyme's abundance will decrease the production of the end product. If the amount of the enzyme is not normally rate-limiting, its presence in a cell must be reduced to rate-limiting levels in order to diminish the pathway's output. Conversely, if the naturally-occurring amount of enzyme is rate limiting, then any increase in the enzyme's activity will result in an increase in the biosynthetic pathway's end product. The modification of nicotine levels in tobacco plants by antisense regulation of putrescence methyl transferase (PMTase) expression is proposed in U.S. Pat. Nos. 5,369,023 and 5,260,205 to Nakatani and Malik. PCT application WO 94/28142 to Wahad and Malik describes DNA encoding PMT and the use of sense and antisense PMT constructs. Additionally, PCT Application WO98/56923 to Conkling et al describes DNA encoding ‘a’ plant quinolate phosphoribosyl transferase (QPRTase) enzyme, constructs comprising such DNA, and methods of altering QPRTase expression to increase or decrease nicotine production in plants. Despite previous efforts and successes, there remains a need for new approaches to reduce the production of gene products in plants (e.g., nicotine).